Inhibitors of Branched-Chain-Aminotransferase-1 (BCAT1) for the Treatment of Brain Tumors

ABSTRACT

Described is a compound capable of reducing or inhibiting (a) the biological activity of branched-chain-aminotrans-ferase-1 (BCATI) or (b) the expression of the gene encoding BCATI for use in a method of treating a neoplasia. A preferred compound is 1-(aminomethyl)cyclohexaneacetic acid (gabapentin).

The present invention provides a compound capable of reducing orinhibiting (a) the biological activity of branched-chainaminotransferase-1 (BCAT1) or (b) the expression of the gen

encoding BCAT1 for use in a method of treating a brain tumor.

Malignant human glioblastomas account for the largest number of humanmalignant brain tumors. So far, the treatment o

gliomas includes neurosurgical techniques (resection o

stereotactic procedures), radiation therapy and chemotherapy The currentstandard of care for, e.g., astrocytic tumor involves surgical tumorresection that can be followed b

chemotherapy with the oral alkylating agent temozolamide (TMZ andradiotherapy. However, glioblastomas are considered a being incurable asthey fail to respond to ionising radiation chemotherapy and surgicalresection. In other words, with these therapies only a very limitedprolongation of the lifespan of patients can be achieved. This meansthat despite these therapies, the average life span after the cancerdiagnosis is merely 12 to 16 months.

Thus, the technical problem underlying the present invention is toprovide means for the therapy of brain tumors preferably glioblastomasor astrocytic brain tumors, which overcome the disadvantages of thecurrent therapies and improve the survival of patients.

The solution of said technical problem is achieved b

providing the embodiments characterized in the claims.

During the experiments resulting in the present invention it was foundthat known BACT1 inhibitors, like gabapentin, which have been used sofar for example as anticonvulsant drugs represent a novel treatmentoption for cancer therapy, i.e. an effective therapy for neoplasias ingeneral and in particular for the treatment of brain tumors likeastrocytic brain tumors. They potentially act by targeting a molecularpathway that is not targeted by any established chemotherapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: IDH^(wt) astrocytic gliomas are characterized by hig

BCAT1 expression.

(a) Schematic representation of BCAA catabolism. BCAAs branched-chainamino acids; BCKAs, branched-chain ketoacids (b,c) RNA expression ofBCAT1 (b) and BCAT2 (c) in 7 astrocytic gliomas (41 IDH^(wt) and 29IDH^(mut)) normalized t

expression in normal brain (dashed line). Data are expressed asmean±s.d. (two-tailed Student's t-test). *, P<0.05; ** P, <0.001. (d)Western blot showing expression of BCAT protein in astrocytic gliomaswith IDH1 and IDH2 wildtype genes (lanes 1-5), different mutations inthe IDH2 (lanes 6-7 or IDH1 (lanes 8-12) genes, and normal brain (lane13). AII diffuse astrocytoma WHO grade II; AAIII, anaplasti astrocytomaWHO grade III; sGBIV, secondary glioblastoma WH grade IV; pGBIV, primaryglioblastoma WHO grade IV; AOIII anaplastic oligodendroglioma WHO gradeIII. (e-h Immunohistochemical stainings of BCAT1 in an IDH^(wt) primaryglioblastoma (e), a primary glioblastoma with IDH1-R132 mutation (f), adiffuse astrocytoma with IDH1-R132C mutation (g), and an anaplasticoligodendroglioma with IDH2-R172 mutation (h). (i,j) Immunohistochemicalstaining of IDH1-R132 in the same tumors as in panels b and c,respectively demonstrating the complementarity of BCAT1 and IDH1-R132staining. Scale bars: 50 μm. (k) Two-by-two table showing thesignificant correlation of BCAT1 protein expression an

mutation status of the IDH1 and IDH2 genes in 81 gliomas (p

0.0001; Fisher's Exact Test).

FIG. 2: BCAT1 shows substrate-dependent expression i

glioblastoma cell lines.

(a) Western blot showing BCAT1 protein expression in IDH glioma celllines. (b-d) Effects of hypoxia and alpha-KG o

BCAT1 expression. RNA and protein expression are shown at the bottom andthe top of each panel. The numbers above the Western blots indicate thefold ratios of expression relative to control cells. The mRNA expressionvalues represent mean s.d. of triplicate samples. (b) BCAT1 isupregulated under hypoxic (1% O₂) conditions. (c) BCAT1 expression isinduced by supplementation of the culture media with cell-permeabledimethyl-alpha KG. (d) Knockdown by two different shRNAs o

the alpha-KG-producing cytoplasmatic IDH1 leads the downregulation ofBCAT1 expression.

FIG. 3: Expression levels of the three BCAT1 transcripts ar associatedwith differential methylation of two alternative promoters.

(a) Schematic drawing of exons 1 to 4 of BCATI showing the exonstructure of the three transcripts T1, T4 and T6. The two alternativepromoters 1 and 2 are shown in the enlarge sections on the lower leftand lower right, respectively. (b

qRT-PCR quantification of RNA expression of transcripts T1, T

and T6 in astrocytic gliomas and a pool of RNAs from normal braintissues (n=23). Values represent mean±s.d. o

triplicate samples. (c) DNA-methylation patterns detected i

promoters 1 (left) and 2 (right) by massarray analysis o

bisulfite-PCR amplicons A1 to A8 in normal brain an astrocytic gliomasWHO grade II-IV. *, IDHwt anaplasti

astrocytoma. (d-f), Extent of DNA methylation in normal brain (Nbr),IDH^(wt) and IDH^(mut) tumors in (d) average of all CpGs i

promoter 2 (two-tailed Student's t-test, P<0.0001) (e) CpG4, (P=0.0003)and (f) CpG6 in promoter 1 (P<0.0001). (g

Correlation of CpG6 methylation grade and BCAT1 T1 expression (h,i)Knockdown of HEY1 in HEK293T cells (h) results in upregulation of BCAT1expression (i). Values represent mean s.d. (n=3). (j,k) ChIP assaysshowing preferred binding o

HEY1 to the DMR (amplicon c1) as compared to control amplicon inpromoter 1 (c2), about 5kb upstream of BCAT1, and unrelate positive(BHLH15) and negative (PTPRD) controls. (j) Flag-HEY construct. (k)HEY1-Flag construct. qRT-PCR was performed i

triplicate and ratios of bound to input are shown a

mean±s.d.

FIG. 4: BCAT1 suppression reduces glutamate release b

glioma cells and affects concentrations of membrane fatt

acids.

(a,b) NMR spectra of U-87MG (a) and U-373MG (b) cells after treatmentwith control (−) or 20 mM gabapentin (+) for 2 hours. Difference spectraare shown near the top of the panels. Upon inhibitor treatment levels ofvaline (Val) leucine (Leu) and isoleucine (Ile) increased by factors1.09 1.38, 1.19, respectively, in U-87MG cells and by factors 1.83 2.18%and 2.32, respectively, in U-373MG cells. (c) Glutamat release by gliomacells at 6 and 12 hours after start o treatment with control (−) or withBCAT1 inhibition (+) by 2 mM gabapentin (n=3). (d) Tandem-MS analysis ofamino acid concentrations in culture media of BCAT1 knockdown andcontrol U-87MG cells 8 days after lentiviral transduction. Values arshown as difference to the media starting concentrations Positive andnegative values indicate amino acid release an

uptake, respectively (n=6). (e) Intracellular amino acid concentrationsof the same cells as in (d) (n=6). (f) Western blot showingdownregulation of HADH upon BCAT1 knockdown. (g

Relative depletion of cholesterol and very long chain fatt

acids in BCAT1 knockdown vs. control U-87MG cells (n=3). Dat

are expressed as mean±s.d. *, P<0.05; **, P<0.01; **

P<0.001.

FIG. 5: BCAT1 knockdown limits glioblastoma cell invasion potential.

(a,b) Immuno-fluorescence labeling of alpha-tubulin in control (a) andBCAT1 knockdown (b) U-87MG cells. blue: DAPI; scal

bar: 50 μm. (c) Sequential images showing the permeation of U-87MG cellthrough a 5×11×300 μm microchannel over a period of 9 hours; scale bar:50 μm. (d) BCAT1 knockdown significantly inhibits the invasion potentialof U-87MG cell compared to nontarget shRNA transduced cells. Resultsindicate the mean±s.d. of three independent experiments. P=0.0146.

FIG. 6: BCAT1 is essential for glioblastoma progression.

(a-d) Cell proliferation and cell cycle analyses of glioma cells uponBCAT1 suppression. Proliferation of cells was examined using theClick-iT® EdU cell proliferation assay Cell cycle analysis was performedafter DNA staining with propidium iodide. The DNA distribution is shownfor living cells. Values in graphs represent mean±s.d. for n=3. ntnontarget shRNA. *, P<0.05; **, P<0.01; *** P<0.001 compared torespective controls. (a) Treatment with the BCAT inhibitor gabapentinfor 20 hours suppressed proliferation of glioma cell lines in aconcentration-dependent manner relative to control cells treated withsolvent only. (b) Cell cycle analysis of gabapentin-treated glioma cellsshowed a accumulation of cells in 01-phase. (c) Knockdown of BCAT causeda significant reduction of cell proliferation relative to samplestreated with nontarget shRNA (nt) in all three glioma cell lines and (d)resulted in the accumulation of the G1-arrest marker CDKN1B/p27^(KIP1).Cell cycle analysis showed significant increases of the proportions ofcells in G1 phase (e) BCAT1 knockdown results in decreasedphosphorylation o

AKT. (f-i) Cross-sections of tumors induced by intracrania injection ofU-87MG glioblastoma cells into CD-1 nude mice Hematoxylin-eosin stainingis shown for mice injected with (f

control nontarget-shRNA or (g) BCAT1-shRNA. (h) Quantification of tumorvolumes (n=5 mice for each group, P=0.0091).

FIG. 7: BCAT1 transcripts.

(a) Gene structure of BCAT1 showing 11 exons and the three transcriptsT1 (ENST00000261192), T4 (ENST00000539282) and T

(ENST00000538118) which originate in two different promoters The regionsaround the transcription start sitzes (TSS) an exon 5 are enlarged toshow primer locations. (b) Agarose ge

image of PCR products amplified from an IDH^(wt) glioblastom (left) anda pool of normal brain RNAs from 23 individual (right) using the reverseprimer and the transcript-specific exon 1 primers. Band sizes match theexpected sizes of the respective spliced mRNAs.

FIG. 8: Western blot analysis

Western Blot analysis of total and phosphorylated protein of the mTORtarget RPS6K upon (a) gabapentin treatment and (b

BCAT1 knockdown in the cell lines U-87MG, U-373MG and Hs683.

FIG. 9: BCAT1 knockdown

BCAT1 knockdown causes apoptosis in a glioblastoma spheroi

primary culture. (a) Reduction of proliferation induced b

lentiviral transduction of two shRNA constructs (n=3, *** P 0.0001). (b)Cell cycle analysis showing strong increase of the subG1 fractionfollowing BCAT1 knockdown (n=3). The Western blot at the top shows theincreased presence of the G1-arrest marker CDKN1B in the knockdowncells. (c

AnnexinV/7AAD assay confirming apoptotic death of spheroi

cells with BCAT1 knockdown.

FIG. 10: Click-iT EdU assay after 5 mM Gabapentin treatment for 23 h

See Example 2 for experimental details.

FIG. 11: Click-iT EdU assay after shRNA-mediated knock down

See Example 3 for experimental details

Thus, the present invention relates to a compound capable of reducing orinhibiting (a) the biological activity o

branched-chain-aminotransferase-1 (BCAT1) or (b) the expression of thegene encoding BCAT1 for use in a method of treating a neoplasia.

“Neoplasm” is an abnormal mass of tissue as a result of neoplasia.“Neoplasia” is the abnormal proliferation of cells The growth of thecells exceeds and is uncoordinated with respect to the normal tissuesaround it. The growth persistant in the same excessive manner, evenafter cessation of the stimuli. It usually causes a lump or tumor.Neoplasms may be benign, pre-malignant (carcinoma-in-situ) or malignant(cancer). The neoplasms to be treated according to the present inventionare those which (over)express BCAT1. Thus, the determination of BCAT1 ina neoplasm is an indication to start with a BCAT inhibiting therapy.Neoplasms to be treated ar

brain tumors, particularly an astrocytic brain tumor, glioma

or glioblastoma, in particular those expressing IDH1 wildtype.

The branched-chain amino acids (BCAAs) valine, leucine an isoleucine areessential amino acids that escape live catabolism and are available inthe general circulation. BCA metabolism provides an important transportsystem to move nitrogen throughout the body for the synthesis ofnon-essential amino acids, including the neurotransmitte glutamate inthe central nervous system. Deregulation of the BCAA catabolic pathwaysfrequently results in neura

dysfunction. The first step of BCAA catabolism involves the transfer ofthe alpha-amino group to alpha-ketoglutarat

(alpha-KG) by the cytosolic branched-chain amino acid transaminase 1(BCAT1) or the mitochondrial BCAT2 isoenzyme with glutamate and therespective branched chain ketoaci

(BCKA) as products (Ichihara et al., J. Biochem. 59, pp. 160 169,(1966); Taylor et al., J. Biol. Chem. 241, pp. 4396-4405 (1966)).Expression of BCAT2 is ubiquitous, whereas expression of BCAT1 isrestricted to a small number of tissues including brain, where BCAAs area major source of nitrogen for the synthesis of the neurotransmitterglutamate. Following transamination, BCKAs are catabolized further toacetyl-Co

and succinyl-CoA, which enter the tricarboxylic acid (TCA cycle (FIG. 1a). NADH and FADH2, which are generated as a by product of BCKAcatabolism, are used to transfer reducing equivalents to complex III ofthe respiratory chain for AT

production.

Mutations in IDH1 (isocitrat dehydrogenase 1), originally detected in afraction of glioblastomas are present in the great majority of WorldHealth Organization (WHO) grade II an

III gliomas and secondary glioblastomas, but are rare in primaryglioblastomas (Bales et al., Acta Neuropath. 116, pp 597-602 (2008)).Mutations in IDH2 also have been detected albeit at a lower frequency of5-10% (Bales, Bales et al. Acta Neuropath. 116, pp. 597-602 (2008); Yanet al., N. Eng

J. Med. 360, pp. 765-773 (2009)). IDH mutations play a central role inglioma pathogenesis (Parsons et al, Science 321, pp 1807-1812 (2008))and have been shown to constitute a ke

classifier distinguishing two major glioma subgroups that wereidentified initially based on RNA expression and DN

methylation patterns. It has been revealed that brain tumor showing anIDH1 mutation (IDH^(mut)) have a better prognosis that those where IDH1is not mutated (IDH^(wt)) (Hartmann et al., Act

Neuropathol. 120, pp. 707-718 (2010)). The inventors found out that onedifference between IDH^(m)

(having IDH1-R132H mutation) and IDH^(wt) brain tumors is that BCAT1overexpression is a highly specific feature of IDH

glioblastomas, the most common and most aggressive adult brain tumor.The observed specific methylation of the BCAT1 promoted in IDH^(mut)tumors, but not in IDH^(wt) tumors and normal brain strongly show thatlow BCAT1 expression in IDH^(mut) tumors is consequence of IDH1mutation-associated DNA methylation, which is thought to be mediatedthrough inhibition of histon

demethylases and the TET family of 5-methylcytosin hydroxylases by theproduct of mutant IDH1 and IDH2 enzymes the oncometabolite2-hydroxyglutarate. Interestingly regulation of BCAT1 mRNA expressionoriginating from promote appears to be achieved by an IDH-independentepigeneti

mechanism, through methylation of a bindung site for the HEYtranscriptional repressor in IDH^(wt) but not IDH^(mut) tumors. Thesediametrically opposed patterns of DNA methylation in the two promotersmake clear that BCAT1 suppression does not occur a

a mere byproduct of IDH1 mutation through “passenger methylation, butrather that the differential regulation of cell metabolism in IDH^(wt)and IDH^(mut) tumors requires tight control of BCAT1 expression in eachgroup.

Similar to the commonly used diagnostic IDH1-R132H staining BCAT1staining may help to distinguish IDH^(wt) from IDH^(m) gliomas; however,BCAT1 staining offers the added advantage c

also distinguishing IDH^(wt) primary glioblastoma from the clos

to 10% of IDH^(mut) astrocytomas with non-IDH1-R132H mutations Mostimportantly, the present invention shows that BCAT1 an

BCAA metabolism provide the basis for the development of nove

metabolism-based approaches in glioma therapy.

This means that suppression of BCAT1 in IDH^(wt) glioblastomas has thepotential to significantly impede tumor growth as well a

the excretion of glutamate by the tumor cells, which frequently causesneurotoxicity to surrounding brain tissue and leads to tumor-associatedepilepsy in brain tumor patients.

In addition, the data of the present invention show that availability oflarge amounts of glucose and glutamine, the two nutrients considered tobe most important for supporting malignant cell growth, is notsufficient to support sustained fast growth of IDH^(wt) glioblastoma.

In other words, in the present invention it has been shown that BCAT1overexpression is a highly characteristic feature of glioblastoma, inparticular IDH^(wt) glioblastoma, and essential for their aggressiveclinical behavior. Thus, BCAT1 expression and BCAA catabolism arepromising markers for the diagnostic and prognostic assessment ofgliomas and serve a

novel therapeutic targets. Furthermore, the present invention representsthe first example of silencing of a metabolic gen

that is central to glioma pathomechanism by IDH1 mutation associatedaberrant DNA methylation. Silencing of BCAT1 earl in tumor developmentwill prevent IDH^(mut) gliomas from utilizing BCAAs as a metabolicresource and offers an explanation for the less malignant growthbehaviour of IDH^(mut) gliomas relative to the BCAT1-dependent IDH^(wt)glioblastomas.

The reduction, silencing or inhibition of the biological activity can beeffected by direct interaction or binding of compound to BCAT1 or byindirect interaction, e.g., b

interacting with a compound that is associated with the biologicalactivity of BCAT1. The reduction or inhibition of the biologicalactivity can also be achieved by the application of altered, e.g.,inactive forms of BCAT1 preferably in excess.

Examples of suitable compounds reducing, silencing o

inhibiting the biological activity of BCAT1 or the expression of thegene encoding BCAT1 with the aim to get a therapeutic effect are:

(a) Plasmids, vectors or natural/synthetic/mutated virusesoligonucleotides of various types of modification (e.g. PTO LNA,2′F-ANA, protein-nucleotide complexes, RNA_(i), siRNA o

mikro_(mi)RNA, shRNA, Methylmethoxy-, Phosphoroamidates, PNA Morpholino,Phosphoramidate, Cyclohexen (CeNA), gap-meres ribozymes, aptamers,CpG-oligos, DNA-zymes, riboswitches, o

lipids or lipid containing molecules;(b) peptides, peptide complexes, including all types of linkers,(c) small molecules;(d) antibodies and their derivatives, especially chimeras Fab-fragments,Fc-fragments, or(e) carriers, liposomes, nanoparticles, complexes, or an other deliverysystems containing the above named constructs,(f) oxidizing agents or sulfhydryl (SH groups) modifying agents.

Further compounds suitable for the purposes of the present invention andmethods how to identify/select such compound are in more detaildescribed below.

Preferably, in a pharmaceutical composition, such compounds as describedabove are combined with a pharmaceuticall acceptable carrier.“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe activ

ingredient and that is not toxic to the host to which it isadministered. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered salin

solutions, water, emulsions, such as oil/water emulsions various typesof wetting agents, sterile solutions etc. Suc

carriers can be formulated by conventional methods and the activecompound can be administered to the subject at an effective dose.

An “effective dose” refers to an amount of the activ

ingredient that is sufficient to affect the course and the severity ofthe neoplasia, leading to the reduction of remission of such apathology. An “effective dose” useful for treating and/or preventingneoplasias may be determined using methods known to one skilled in theart (see for example Fingl et al., The Pharmocological Basis ofTherapeutics Goodman and Gilman, eds. Macmillan Publishing Co., New Yorkpp. 1-46 ((1975)).

Administration of the suitable compositions may be effected b

different ways, e.g. by intravenous, intraperitoneal subcutaneous,intramuscular, topical or intraderma

administration. The route of administration, of course depends on thekind of therapy and the kind of compound contained in the pharmaceuticalcomposition. The dosage regimen will be determined by the attendingphysician and other clinical factors. As is well known in the medicalarts dosages for any one patient depends on many factors, including thepatient's size, body surface area, age, sex, th

particular compound to be administered, time and route o

administration, the kind of therapy, general health and of the drugsbeing administered concurrently.

The person skilled in the art can easily identify or generate compoundsuseful for the treatments of the present invention based on theknowledge of the amino acid sequence of BCAT1 and the nucleotidesequence of the gene encoding this protein Respective sequences arefound in the UniProtKB/Swiss-Pro

database (P54687; BCAT1 HUMAN), in Genbank (NCBI Reference Sequence:NM_(—)005504) and the Human Genome Organization Gen

Nomenclature Committee (HGNC) database (HGNC ID: 976).

In a further preferred embodiment of the present invention the compounduseful for reducing or inhibiting the expression of the gene encodingBCAT1 is an antisense oligonucleotide shRNA or siRNA specific for BCAT1.Preferably, the compound useful for silencing the BCAT1 expression areindependent Mission® shRNA constructs targeting different regions of thhuman BCAT1 (BCAT1 shRNAI NM_(—)005504.3-1064s1c1 and BCAT shRNAIINM_(—)005504.3-751s1c1) and human IDH1 (IDH1 shRNANM_(—)005896.2-1363s1c1 and IDH1 shRNAII NM_(—)005896.2-292s1c1 mRNAtranscripts.

The generation of suitable antisense oligonucleotides includedetermination of a site or sites within the BCAT1 encoding gene for theantisense interaction to occur such that the desired effect, e.g.,inhibition of the expression of the protein, will result. A preferredintragenic site is (a) the region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of thegene or (b)

region of the mRNA which is a “loop” or “bulge”, i.e., no

part of a secondary structure. If one or more target site have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridiz

sufficiently well and with sufficient specificity, to give the desiredeffect. In the context of this invention “hybridization” means hydrogenbonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding between complementary nucleoside or nucleotide bases

“Complementary” as used herein, refers to the capacity for precisepairing between two nucleotides. For example, if

nucleotide at a certain position of an oligonucleotide i

capable of hydrogen bonding with a nucleotide at the same position of aDNA or RNA molecule, then the oligonucleotide and the DNA or RNA areconsidered to be complementary to each other at that position. Theoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can make hydrogen bonds with each otherThus, “specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity or precisepairing such that stable an

specific binding occurs between the oligonucleotide and the DNA or RNAtarget. It is understood in the art that the sequence of an antisensecompound does not need to be 100 complementary to that of its targetnucleic acid to be specifically hybridizable. An antisense compound i

specifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-targe sequences under conditions in which specific binding i

desired, i.e., in the case of therapeutic treatment.

The skilled person can generate antisense compounds and siRNA or shRNAsaccording to the present invention on the basis of the known DNAsequence for BCAT1.

“Oligonucleotide” refers to an oligomer or polymer o

ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) o

mimetics thereof. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well a

oligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhance

cellular uptake, enhanced affinity for nucleic acid target an increasedstability in the presence of nucleases. While antisense oligonucleotidesare a preferred form of the antisense compound, the present inventioncomprehends other oligomeric antisense compounds, including but notlimited to oligonucleotide mimetics such as are described below. Theantisense compounds in accordance with this invention compris

from about 8 to about 50 nucleobases (i.e. from about 8 t about 50linked nucleosides). Particularly preferred antisense compounds areantisense oligonucleotides, even more preferable those comprising fromabout 15 to about 25 nucleobases Antisense compounds include ribozymes,external guide sequences (EGS), oligonucleotides (oligozymes), and othershort catalytic RNAs or catalytic oligonucleotides which hybridize tothe target nucleic acid and inhibit it

expression.

Alternatively, the compound of the invention is a vector allowing totranscribe an antisense oligonucleotide of the invention, e.g., in amammalian host. Preferably, such vector is a vector useful for genetherapy. Preferred vector useful for gene therapy are viral vectors,e.g. adenovirus herpes virus, vaccinia, or, more preferably, an RNAvirus suc

as a retrovirus. Even more preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples o

such retroviral vectors which can be used in the presen invention are:Moloney murine leukemia virus (MoMuLV), Harve

murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV) andRous sarcoma virus (RSV). Most preferably, a non-human primateretroviral vector is employed, such as th gibbon ape leukemia virus(GaLV), providing a broader hos

range compared to murine vectors. Since recombinan retroviruses aredefective, assistance is required in order to produce infectiousparticles. Such assistance can be provided e.g., by using helper celllines that contain plasmid encoding all of the structural genes of theretrovirus unde

the control of regulatory sequences within the LTR. Suitable helper celllines are well known to those skilled in the art Said vectors canadditionally contain a gene encoding selectable marker so that thetransduced cells can b

identified. Moreover, the retroviral vectors can be modified in such away that they become target specific. This can be achieved, e.g., byinserting a polynucleotide encoding sugar, a glycolipid, or a protein,preferably an antibody Those skilled in the art know additional methodsfor generating target specific vectors. Further suitable vector andmethods for in vitro- or in vivo-gene therapy ar

described in the literature and are known to the person skilled in theart; see, e.g., WO 94/29469 or WO 97/00957.

In order to achieve expression only in the target organ, e.g. a braintumor, the DNA sequences for transcription of the antisenseoligonucleotides can be linked to a tissue specific promoter and usedfor gene therapy. Such promoters are well known to those skilled in theart (see e.g. Zimmermann et al. (1994) Neuron 12, 11-24; Vidal et al.;(1990) EMBO J. 9, 833 840; Mayford et al., (1995), Cell 81, 891-904;Pinkert et al. (1987) Genes & Dev. 1, 268-76).

Within an oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbon of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage. Specific examples of preferred antisensecompounds useful in the present invention include oligonucleotidescontaining modified backbones or non-natural internucleoside linkagesOligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. Modified oligonucleotide backbones which canresult in increased stability are known to the person skilled in the artpreferably such modification is a phosphorothioate linkage.

A preferred oligonucleotide mimetic is an oligonucleotide mimetic thathas been shown to have excellent hybridizatio properties, and isreferred to as a peptide nucleic aci

(PNA). In PNA compounds, the sugar-backbone of a oligonucleotide isreplaced with an amide containing backbone in particular anaminoethylglycine backbone. The nucleobase are retained and are bounddirectly or indirectly to az

nitrogen atoms of the amide portion of the backbone (see e.g., Nielsenet al., Science 254 (1991), 1497-1500.)

Modified oligonucleotides may also contain one or more substituted ormodified sugar moieties. Preferred oligonucleotides comprise one of thefollowing at the 2 position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O- S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ t C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. A particularly preferred modifiedsugar moiety is a 2′-O-methoxyethyl suga

moiety.

Oligonucleotides of the invention may also include nucleobasmodifications or substitutions. Modified nucleobases include othersynthetic and natural nucleobases such as 5 methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine hypoxanthine, 2-aminoadenine,6-methyl and other alky

derivatives of adenine and guanine, 2-propyl and other alky

derivatives of adenine and guanine, 2-thiouracil, 2 thiothymine and2-thiocytosine etc., with 5-methylcytosin

substitutions being preferred since these modifications have been shownto increase nucleic acid duplex stability.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include lipid moieties suchas a cholesterol moiety cholic acid, a thioether, a thiocholesterol, analiphati

chain, e.g., dodecandiol or undecyl residues, a phospholipid

a polyamine or a polyethylene glycol chain, or adamantan

acetic acid, a palmityl moiety, or an octadecylamine o

hexylamino-carbonyl-oxycholesterol moiety.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds o

“chimeras,” in the context of this invention, are antisens compounds,particularly oligonucleotides, which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of a oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide i

modified so as to confer upon the oligonucleotide increase resistance tonuclease degradation, increased cellular uptake and/or increased bindingaffinity for the target nucleic acid An additional region of theoligonucleotide may serve as substrate for enzymes capable of cleavingRNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellularendonucleas

which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNaseH, therefore, results in cleavage of the RNA target thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotide when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Chimeric antisense compounds of the invention may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleoside and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids o

gapmers.

In a further preferred embodiment of the present invention the compoundsfor use in a method of treating a neoplasia are compounds reducing orinhibiting the biological activity o

BCAT1.

Such compounds are described in the art for medica

indications differing from the indication of the present invention and,preferably, comprise compounds like 1 (aminomethyl)cyclohexaneaceticacid (gabapentin) or a compound described in Goto et al., The Journal ofBiological Chemistry 280(44) (2005), 37246-56, Hu et al., Bioorganic &Medicinal Chemistry Letters 16 (2006), 2337-40, Caballero et al.Molecular Diversity 13 (2009), 493-500, U.S. Pat. No. 6,632,831, U.S.Pat. No. 6,809,119, and EP-B1 1 157 000.

Further examples of compounds capable of reducing o

inhibiting the biological activity of BCAT1 are (neutralizing antibodiesdirected against BCAT1 or fragments thereof having substantially thesame binding specificity. The term “antibody”, preferably, relates toantibodies which consists essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations. Monoclonal antibodies are made from a antigencontaining, e.g., a fragment of BCAT1 by methods well known to thoseskilled in the art (see, e.g., Köhler et al. Nature 256 (1975), 495). Asused herein, the term “antibody

(Ab) or “monoclonal antibody” (Mab) is meant to include intact moleculesas well as antibody fragments (such as, for example Fab and F(ab′).2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the F

fragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody.(Wahl et al., J. Nucl. Med. 24: 316 325 (1983)). Thus, these fragmentsare preferred, as well a

the products of a FAB or other immunoglobulin expression library.Moreover, antibodies useful for the purposes of the present inventioninclude chimerical, single chain, an

humanized antibodies.

Alternatively, preferred compounds for the purpose of the invention areinactive versions of BCAT1 or nucleic acid sequences encoding inactiveversions of BCAT1 that can be introduced according to theapproaches/vectors describe above. Such inactive versions can begenerated according to well known methods of mutagenesis. Such compoundscan have therapeutic effect in the human body by displacing theirfunctionally active counterpart, in particular when applied i

excess. Analyses of potentially inactive versions of BCAT1 can becarried out by assaying the (reversible) transamination o

branched-chain L-amino acids to branched-chain alpha-ket

acids, e.g., by determining the production of glutamate

Suitable assays are described in the literature and, e.g., i

Example 39 of U.S. Pat. No. 6,809,119, Hu et al. (2006), an EP-B1 1 157000.

In a preferred embodiment of the present invention the compounddescribed in detail above is used in a method o

treating an astrocytic brain tumor or glioblastoma.

The present invention also relates to a method for identifying acompound reducing or inhibiting the biological activity o

BCAT1 and/or its expression, comprising the steps of:

-   -   (a) incubating a candidate compound with a test system        comprising BCAT1 or its gene; and    -   (b) assaying a biological activity of BCAT1;    -   wherein an inhibition or loss of a biological activity o        BCAT1, preferably compared to a test system in the absence of        said test compound, is indicative of the presence of        candidate compound having the desired property.

Step (b) can be carried out by assaying the (reversible transaminationof branched-chain L-amino acids to branched chain alpha-keto acids usingan assay as described above.

Examples of such candidate molecules include antibodiesoligonucleotides, proteins, or small molecules. Such molecule can berationally designed using known techniques.

Preferably, said test system used for screening comprise substances ofsimilar chemical and/or physical properties most preferably saidsubstances are almost identical. The compounds which can be prepared andidentified according to use of the present invention may be expressionlibraries e.g., cDNA expression libraries, peptides, proteins, nucleicacids, antibodies, small organic compounds, ligands, hormones

peptidomimetics, PNAs or the like.

WO 98/25146 describes further methods for screening libraries ofcomplexes for compounds having a desired property especially, thecapacity to agonize, bind to, or antagonize polypeptide or its cellularreceptor. The complexes in such libraries comprise a compound undertest, a tag recording a least one step in synthesis of the compound, anda tethe

susceptible to modification by a reporter molecule Modification of thetether is used to signify that a comple

contains a compound having a desired property. The tag can be decoded toreveal at least one step in the synthesis of such compound. Othermethods for identifying compounds which interac

with BCAT1 or nucleic acid molecules encoding such molecule are, forexample, the in vitro screening with the phage display system as well asfilter binding assays or “real time” measuring of interaction.

It is also well known to the person skilled in the art, that it ispossible to design, synthesize and evaluate mimetics o

small organic compounds that, for example, can act as substrate orligand to BCAT1. For example, it has been described that D-glucosemimetics of hapalosin exhibited similar efficiency as hapalosin inantagonizing multidru

resistance assistance-associated protein in cytotoxicity; se

Dinh, J. Med. Chem. 41 (1998), 981-987.

All these methods can be used in accordance with the present inventionto identify a compound reducing or inhibiting the biological activity ofBCAT1 or its expression.

The gene encoding BCAT1 can also serve as a target for the screening ofinhibitors. Inhibitors may comprise, for example proteins that bind tothe mRNA of the genes encoding BCAT1 thereby destabilizing the nativeconformation of the mRNA an hampering transcription and/or translation.Furthermore methods are described in the literature for identifyingnucleic acid molecules such as a RNA fragment that mimics the structureof a defined or undefined target RNA molecule t

which a compound binds inside of a cell resulting in the retardation ofthe cell growth or cell death; see, e.g., W

98/18947 and references cited therein. These nucleic acid molecules canbe used for identifying unknown compounds o

pharmaceutical interest, and for identifying unknown RNA targets for usein treating a disease. These methods an

compositions can be used for identifying compounds useful to reduceexpression levels of BCAT1.

The compounds which can be tested and identified according to the methodof the invention may be expression libraries, e.g. cDNA expressionlibraries, peptides, proteins, nucleic acids antibodies, small organiccompounds, hormones peptidomimetics, PNAs or the like (Milner, NatureMedicine (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 7

(1994), 193-198 and references cited supra). Furthermore genes encodinga putative regulator of BCAT1 and/or which exert their effects up- ordownstream of BCAT1 may be identified using, for example, insertionmutagenesis using for example, gene targeting vectors known in the art.Sai

compounds can also be functional derivatives or analogues o

known inhibitors. Such useful compounds can be for example transactingfactors which bind to BCAT1 or regulator sequences of the gene encodingit. Identification o

transacting factors can be carried out using standard method in the art.To determine whether a protein binds to the protein itself or regulatorysequences, standard native gel

shift analyses can be carried out. In order to identify transactingfactor which binds to the protein or regulator sequence, the protein orregulatory sequence can be used as a affinity reagent in standardprotein purification methods, o

as a probe for screening an expression library. Th

identification of nucleic acid molecules which encod polypeptides whichinteract with BCAT1 can also be achieved, for example, as described inScofield (Science 274 (1996), 2063 2065) by use of the so-called yeast“two-hybrid system”. In this system BCAT1 is linked to the DNA-bindingdomain of the GAL transcription factor. A yeast strain expressing thisfusio

polypeptide and comprising a lacZ reporter gene driven by a appropriatepromoter, which is recognized by the GAL transcription factor, istransformed with a library of cDNA which will express plant proteins orpeptides thereof fused t

an activation domain. Thus, if a peptide encoded by one of the cDNAs isable to interact with the fusion peptide comprising peptide of BCAT1,the complex is able to direct expression o

the reporter gene. In this way, BCAT1 and the gene encoding BCAT1 can beused to identify peptides and proteins interacting with BCAT1. It isapparent to the person skilled in the art that this and similar systemsmay then further be exploited for the identification of inhibitors.

The below example explains the invention in more detail bu

are not construed as a limitation of the invention.

Example 1 General Methods

(a) RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted using the AllPrep DNA/RNA/Protein Min

Kit (Qiagen) according to the manufacturer's instructions FirstChoice®Human Brain Reference Total RNA from Ambio

served as normal brain RNA pool (n=23 donors). Total RNA (50 ng) wasreverse-transcribed using random primers an superscript II (Invitrogen,Karlsruhe, Germany) according to the manufacturer's instructions. Eachcomplimentary DNA sample was analysed in triplicate with the AppliedBiosystems Pris

7900HT Fast Real-Time PCR System (Applied Biosystems, Foste

City, Calif., USA) using Absolute SYBR Green ROX Mix (ABgene

Epsom, UK). The relative amount of specific mRNA was normalised to ARF,B2M and TBP. Primer sequences are given in the following Table 1A.

TABLE 1A Expression primer BCAT1 Forward CAACTATGGAGAATGGTC(all isoforms) CTAAGCT Reverse TGTCCAGTCGCTCTCTTC TCTTC BCAT1 T1 ForwardGCTACGACCCTTGGGATC (ENST00000261192) T BCAT1 T4 ForwardGTGCCACTGCCGCTCTCT (ENST00000539282) BCAT1 T6 Forward TGGTTGTCTGAGCCTCCT(ENST00000538118) TT BCAT1 Exon 2 Reverse AAGTCCCCACCACCTCTT TTBCAT1 Exon 5 Reverse CCCATTCTTGATCCAATT TCA HEY1 ForwardCGAGCTGGACGAGACCAT Reverse GAGCCGAACTCAAGTTTC CA ARF ForwardGACCACGATCCTCTACAA GC Reverse TCCCACACAGTGAAGCTG ATG B2M ForwardACTGAATTCACCCCCACT GA Reverse CCTCCATGATGCTGCTTA CA TBP ForwardGAACCACGGCACTGATTT TC Reverse CCCCACCATGTTCTGAAT CT ReverseAAATAACTTAACACCCCA ATCTAAAC

(b) Western Blot Analysis

All experiments involving the use of human tissues are carried out inline with the guidelines of the institutional revie

board of the Medical Faculty at Heinrich Heine University Düsseldorf.Fresh frozen human tumor tissue samples and a non-neoplastic braintissue sample were lysed in guanidin isothiocyanate solution (4M) usingan ULTRA-TURRAX (IKA Staufen, Germany) and subjected toCsCl-ultracentrifugation Diafiltration of the obtained protein fractionswas performed using the Amicon Ultra-0.5 centrifugal filter device(Millipore, Schwalbach, Germany; 3 kDa cut-off) essentially adescribed⁴⁵. Total protein of cell lines was extracted using the AllPrepDNA/RNA/Protein Mini kit (Qiagen). 10 μg o

protein were separated by 10% SDS-PAGE and transferred to PVD membranes(Millipore). The membrane was blocked in blocking solution (5% BSA inTBS, 0.1% Tween 20) and incubated with primary antibodies overnight at4° C. Horseradish peroxidas

(HRP)-conjugated secondary antibodies were incubated for hour at roomtemperature prior to the chemiluminescent detection of protein (ECL kit;GE Healthcare, Little Chalfont

UK).

The following antibodies were used: monoclonal mouse antibod

against α-tubulin (1:2000, #T9026, Sigma-Aldrich, St. Louis Mo. 63178,USA), monoclonal mouse antibody against BCAT (1:2000, ECA39, clone 51,#611270, BD Biosciences, San Jose Calif., USA), monoclonal rat antiserumagainst IDH1 (1:1, provided by A. von Deimling, DKFZ, Heidelberg,Germany; commercially available from Dianova, Hamburg, Germany),monoclonal mous

antibody against HADHSC (1:500, #H00003033-M01, Abnova) monoclonalrabbit antibody against CDKN1B (1:1000, p27 Kip1 #3686, Cell SignalingTechnology), monoclonal rabbit antibod against Akt (1:1000, #9272, CellSignaling Technology) monoclonal rabbit antibody against phospho-Akt(Ser473 (1:1000, #193H12, Cell Signaling Technology), polyclona rabbitantibody against p70 S6 kinase (1:1000, #9202, Cell SignalingTechnology), polyclonal rabbit antibody against phospho-p70 S6 kinase(Thr389) (1:1000, #9205, Cell Signaling Technology), HRP-conjugatedanti-rat IgG (1:10000, provided by A. von Deimling, DKFZ, Heidelberg,Germany), HRP-conjugated horse anti-mouse IgG (1:5000, #7076, CellSignaling Technology), HRP-conjugated goat anti-rabbit IgG (1:2000#7074, Cell Signaling Technology).

(c) Immunohistochemical Staining

Tumor sections were deparaffinised using xylol and rehydrated indecreasing concentrations of ethanol. Antigen retrieval was performed byheating for 40 min in a steamer in 10 mM sodium citrate buffer (pH 6.0).Endogenous peroxidase was inactivate by incubating the tumor sections in3% hydrogen peroxide Sections were incubated overnight with primaryanti-BCAT (ECA39) monoclonal mouse antibody, clone 51 (BD BiosciencesSan Jose, Calif.) diluted 1:500 or mouse anti-human IDH1 R132 antibody(Dianova, Hamburg, Germany) diluted 1:20 in Dak

REAL™ antibody diluent (Dako, Glostrup, Denmark). Staining for detectionof bound antibody was performed according to standard protocols usingthe EnVision™ detection system (peroxidase/DAB+, rabbit/mouse) (Dako,Glostrup, Denmark) subsequent counterstaining was done usinghematoxylin.

(d) DNA Methylation Analysis

DNA methylation was analyzed by MassARRAY technique. Briefly 500 nggenomic DNA was chemically modified with sodium bisulfite using the EZmethylation kit (Zymo Research according to the manufacturer'sinstructions. The bisulfite

treated DNA was PCR-amplified with primers generating fou

amplicons (A1-A4) from −990 bp to +612 bp around TSS of BCAT transcript1 (T1, ENST00000261192), and three amplicons (A5 A7) of the promoter ofBCAT1 transcript 4 (T4 ENST00000539282) and 6 (T6, ENST00000538118) from−198 bp t

+63 bp. The amplicons were transcribed by T7 polymerase followed by Tspecific-RNAaseA cleavage. The digested fragments were quantified bymass-spectrometry. The prime sequences are given in Table 1B. DNAmethylation standard (0%, 20%, 40%, 60%, 80%, and 100% methylatedgenomic DNA) were used to confirm the unbiased amplification of theamplicons.

TABLE 1B Methylation analysis primer A1 * ForwardAAGTTTTTGTTGTGGAAGTTGTTTT Reverse CACCTAACCAACAATCATTAAACACTA A2 *Forward TTTGTTTGAGGGTATTGGAAGTAAT Reverse TAACTCCTACCCACCTCCCTACTAT A3 *Forward ATAGTAGGGAGGTGGGTAGGAGTTA Reverse AAACACTAAAACTACTCCCCCAAAC A4 *Forward GTTTGGGGGAGTAGTTTTAGTG Reverse CTCCCTACCAACTATATTTCTTA A5 *Forward ATTTATGAGGGTGTTAGATTTGGGT Reverse TTAAAAACTCCTCCCCAAAAAATAC A6 *Forward TTGTTTAGGTTTAGTATTTTTATGGG Reverse ACCATTTATAAAAAAATCTCCAAAAA7 * Forward AAATTATTATTAAGTAAATGTAGGTGGG

(e) Cell Culture

The human glioma cell lines U-87MG (HTB-14), LN-229 (CRL 2611), Hs683(HTB-138) and U-373MG (HTB-17) were cultured i

Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetalcalf serum and 1% penicillin/streptomycin-mix. Cel lines wereauthenticated by short tandem repeat (STR analysis. The brain cancerstem-like cells NCH421k were established from primary glioblastomapatients undergoing surgical resection according to the researchproposal approved by the institutional review board at the Medica

Faculty, University of Heidelberg. They were characterized genotypicallyand phenotypically in a previous study (Campos

B., et al. Differentiation therapy exerts antitumor effects o

stem-like glioma cells. Clin Cancer Res 16, 2715-2728 (2010)) NCH421kcells were grown as floating aggregates (neurospheres

on uncoated tissue culture dishes in DMEM/F-12 mediu

containing 20% BIT serum-free supplement, basic fibroblas

growth factor and epidermal growth factor at a concentration of 20 ng/mleach (all from Provitro, Berlin, Germany). HEK293 and HEK293 cells weremaintained as monolayer cultures in DME

containing 10% fetal calf serum without antibiotics.

All cell lines were cultivated at 37° C. in a humidified incubator with5% CO₂. For hypoxia experiments, cells were cultured at 1% O₂ in anitrogen-supplied C16 hypoxia incubato

(Labotect, Goettingen, Germany).

(f) Chromatin Immunoprecipitation (ChIP)

ChIP was performed on HEK293 cells overexpressing flag-tagged HEY1,since the currently available antibodies against HEY were not specificfor ChIP assays. Constructs for HEY overexpression were prepared usinggateway compatible vector tagged with FLAG; pDest26 (C-terminal tag) andpDest11 (N

terminal tag). 1 μg of either the control vectors or HEY expressionvectors were transfected into 2.5×10̂6 HEK293 cell per 15 cm plate usingTransIT®-LT1 (Mirus Bio LLC, Madison, W

53711, USA) according to slightly modified manufacturer' instructions.The cells from two plates were harvested 4 hours after transfection.Chromatin was prepared using non

ionic shearing buffers with Covaris S2, according to the manufacturer'sprotocol (Covaris Inc.). ChIP was performed using anti-FLAG antibody(Cell signaling #2368). The DNA recovered after ChIP was used forqRT-PCR with input chromati

and mock immunoprecipitation (anti-IgG antibody, Diagenode Kch-803-015)serving as controls. qRT-PCR was performed i

triplicate with SYBR green detection using primers listed i

Table 2. Ratios of bound to input signals are reported.

TABLE 2 Promoter primer PTPRD Forward GAGGAGGAGGAGAAAGAGCA ReverseGACAGAGCCTCGCCTCCT BCAT1_neg Forward TCCCTAGTCCTCCGTTCTGA ReverseATTCCAAGGAGCATTTGTGC BCAT1_DMR(c1) Forward GAGGGTGACTAAGGAACTCTGGReverse ATTGCCATTCCGTCATCACT BCAT1_c2 Forward GCTACGACCCTTGGGATCTReverse TCGATTCACGCACACATTTT BHLHA15 Forward CCGAGGGCTCATTTGCAT ReverseCACCCGAGTTCCCAGCTC

(g) Transient Transfection of siRNA

HEK293T cells were transfected with siRNA duplexes from Ambio

(HEY1: s23868-70) or Dharmacon (control non-targeting: D

001810-10) using DhamaFect1 following slight modifications t

the manufacturer's instructions (Dharmacon). Briefly, eac

siRNA pool was diluted in 1× siRNA buffer (Dharmacon), wa

mixed with RPMI, and then distributed into 6 wells of 96 wel

plates. 1.2×10̂4 HEK293 or 9×10̂3 HEK293T cells were seeded o top of thesiRNA/DharmaFECT mixture (the volume was 15 μl/well and 20 nM of siRNAsin final). After 48 hour incubation at 37° C., RNAs were isolated fromthe wells fo

further analysis.

(h) Virus Production and Transduction

Lentiviral vectors were produced by cotransfection of HEK293 cells withthe psPAX2 (Addgene 12260, Didier Trono, packaging vector), pMD2.G(Addgene 12259, Didier Trono, envelop

plasmid), and pLKO.1 shRNA constructs (Sigma-Aldrich) Transfections werecarried out using TransIT®-LT1 (Mirus Bi

LLC) and virus was harvested at 48 and 72 hours after transfection andcombined.

Infection of glioma cells with virus at an M.O.I. of 2 was carried outin the presence of 8 μg/ml of polybrene (Chemicon Billerica, Mass.,USA). Virus-containing supernatant was removed after 24 hours and cellswere split on day 3, day 5 and day

after transduction. Two independent Mission® shRNA construct

targeting different regions of the human BCATI (BCAT1 shRNANM_(—)005504.3-1064s1c1 and BCAT1 shRNAII NM_(—)005504.3-751s1c1 andhuman IDH1 (IDH1 shRNAI NM_(—)005896.2-1363s1c1 and IDH

shRNAII NM_(—)005896.2-292s1c1) mRNA transcripts were used NontargetingshRNA (Mission SHC002) was used as a control. Quantification of BCAT1and IDH1 knockdown was assessed b

quantitative real-time PCR and Western blot.

(i) Treatment with Dimethyl-α-Ketoglutarate and Gabapentin

5×10̂4 cells/well were seeded in 24-well plates in a total volume of 500μl cell culture medium. The medium was removed 16 hours after seedingthe cells, and replaced with 500μ medium containing 5 mM or 10 mMdimethyl-α-ketoglutarat

(Dimethyl 2-oxoglutarate; Sigma-Aldrich) or 5 mM, 10 mM or 2 mMgabapentin (1-(Aminomethyl)-cyclohexane; Sigma-Aldrich) Correspondingvolumes of 200 mM HEPES buffer (pH 7.4) were added to the respectivecontrol wells.

(j) Cell-Cycle Analysis and Detection of Apoptosis

Cell-cycle analysis was performed 20 hours after treatment withgabapentin and 6 days after lentiviral transduction Nicoletti buffer(0.1% sodium citrate, pH7.4, 0.05% Triton X 100, 50 μg ml⁻¹ propidiumiodide) was added to the well

containing both dead and living cells. After 4 hours in the dark at 4°C., DNA content was analysed by flow cytometry using FACS Canto II (BDBiosciences, San Jose, Calif., USA). FACS Div

software was used to quantify the distribution of cells i

each cell-cycle phase: sub-G1 (dead cells), G1, S, and G2/M. Forinvestigation of apoptotic activity after lentivira

knockdown NCH421k cells were incubated with annexin V-P (Phycoerythrin)and 7-AAD (7-aminoactinomycin D, B

Biosciences) for 15 minutes in the dark, immediately followed by flowcytometry.

(k) Proliferation Analysis

To assess the proliferation of glioma cells after treatment withgabapentin or after lentiviral knockdown, the Click-iT

EdU cell proliferation assay (Invitrogen, Karlsruhe, Germany was usedfollowing the manufacturer's instructions. The cell were incubated with10 μM of the nucleoside analog EdU (5 ethynyl-2′deoxyuridine) for 16hours. Quantification of cell that incorporated EdU was performed usingFACS Canto II (B

Biosciences).

(l) Glutamate Quantification

Glutamate concentration in the supernatant of cells treated withgabapentin was determined using the glutamine/glutamat

determination Kit (GLN-1; Sigma-Aldrich) according to the manufacturer'sinstructions. The reaction volumes were scaled down to 100 μl totalvolume and absorbance was measured in triplicate in a microplate(Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate) using aTecan Infinite M200 plat

reader (Tecan, Austria). Data were normalised to the number o

cells per well.

(m) Immunofluorescence Staining

Cells were plated on glass coverslips five days after lentiviraltransduction. Cells were fixed in 4% formaldehyde rinsed twice in 1×PBS,and permeabilized in PBS containing 0.2% Triton. Following rinsing with1×PBS cells were incubated in 10% goat serum for 30 minutes at roomtemperature. Samples were incubated 1 hour with the primary antibody(mouse anti α-tubulin antibody, 1:200, #T9026, Sigma Aldrich) and 1 hourat room temperature with the secondary antibody (FITC-conjugated goatanti mouse antibody, 1:100 ab6785, Abcam) following Mounting withDAPI-containing Vectashield mounting medium (Vector Laboratories,Burlingame Calif., USA). For fluorescence imaging, images were takenusing 40× objective lense on a Zeiss Axioplan microscope.

(n) 3D Microchannel Migration Assay

Poly(dimethylsiloxane) (PDMS) based microchannel chips were kindlyprovided by Dr. Ralf Kemkemer (Max Planck Institute for IntelligentSystems, Germany). Microfabricated channe structures with the dimensionsof 5×11×300 μm (W×H×L) were bio-functionalized by incubation with a 50μg/ml fibronecti

solution prior to use. The chip was fixed on a Teflon holde

and 2×10̂5 cells were seeded on the chip in close proximity t

the channels. After cells were attached on the chip, live-cell imagingwas performed for 25 hours. During the experiments n

chemical gradient or flow inside the channels was applied Phase-contrasttime-lapse pictures of multiple positions were captured every 10 minuteswith an automated inverted microscope (Zeiss Cell Observer; Carl Zeiss)equipped with a

air-humidified and heated chamber. Images were recorded an

processed with Zeiss AxioVision and ImageJ software. Cel

behaviors were analyzed and categorized as reported previously (Bai, A.H., et al. MicroRNA-182 promotes leptomeningeal sprea

of non-sonic hedgehog-medulloblastoma. Acta Neuropatho (2011); Rolli, C.G., Seufferlein, T., Kemkemer, R. & Spatz

J. P. Impact of tumor cell cytoskeleton organization o

invasiveness and migration: a microchannel-based approach PLoS One 5,e8726 (2010)).

(o) Animal Experiments

Animal work was approved by the governmental authorities(Regierungspraesidium Karlsruhe, Germany) and supervised b

institutional animal protection officials in accordance with theNational Institutes of Health guidelines Guide for the Care and Use ofLaboratory Animals.

(p) Orthotopic Brain Tumor Model

A total of 2×10̂5 U-87MG cells with BCAT1 shRNAI knockdown o

nontargeting shRNA were stereotactically implanted into the brain of six7-9-week-old athymic mice (CD1 nu/nu; Charle

River Laboratories, Wilmington, Mass., USA), respectively. Fou

weeks after implantation, animals were sacrificed, brain removed andcryosectioned. Brain sections were stained with hematoxylin and eosinand the tumor volume was calculate using ImageJ.

(q) Perchloric Acid Cell Extraction and NMR Spectroscopy

Cells were harvested by trypsinization, washed once with ice cold PBS,and the cell pellet was frozen at −80° C. Typically 1×10⁸ harvestedcells were subjected to perchloric acid extraction followed by KOHneutralization according t

published protocols. Briefly, 2 mL of ice-cold 1 N HClO₄ were added to afrozen pellet of harvested cells. The pellet was disrupted using aTeflon pestle and a glass homogeniser. 1 m

of ice-cold water was added to the lysate, vortexed for 90

and centrifuged at 10000×g for 10 min at 4° C. The pellet wasre-extracted and the supernatants were combined. The extrac

was neutralised with KOH and the pH was adjusted to 6.5-7.0

Precipitated KClO₄ was pelleted at 25000×g for 20 min and thesupernatant was lyophilised.

The lyophilised extract residues were dissolved in 0.5 ml of D₂O buffer(99.9% D) containing 30 mM sodium phosphate and 0. mM sodium azide (pH7.02). For quantitative analysis reference capillary (1.5 mm OD, 0.99 mmID) was filled with solution of 11 mMtrimethylsilyl-2,2,3,3-tetradeuteropropanoi

acid (TSP, sodium salt) in the D₂O buffer described above Subsequently,the capillary was sealed in the flame of Bunsen burner. A calibrationsolution was prepared by placin weighed amounts of glucose and citricacid in the D₂O buffer t

give the calculated concentrations of 10.49 mM glucose an 4.95 mmglucose. ¹H-NMR spectra of the calibration solution capillary wereacquired at 600 MHz (Avance AV-600, Bruke

BioSpin GmbH) using a standard 5-mm NMR tube and a triple resonanceinverse probe under the same conditions to be use for the extracts (20°C., presaturation of residual HDO signal repetition time TR=7 s, 30°flip angle). The signal integrals for citrate and glucose were set tovalue corresponding to the concentrations given above, and th

integral of the TSP methyl signal was found to be equivalen

to 4.60±0.10 mM protons. Cell extracts were measured wit

the calibrated reference capillary inserted (512 transients i

1 hour), and the ¹H-NMR signals for the branched-chain amin

acids, various metabolites, and the TSP reference wer

integrated. The TSP integral was defined as 4.60 mM so tha

the metabolite signal integrals gave directly the metabolit

concentrations in mM (μmol/ml) for the 0.5 ml volumes used These datawere then converted to femtomol/cell using the cel

counts determined prior to extraction.

(r) Preparation of Cell Homogenates

Cell pellets were diluted in 100 μl water and homogenized b.sonification (Ultrasonic device, 3−5×20 cycle, output 80% BransonSonifier 450, Dietzenbach, Germany). Protein wa

determined according to Lowry (Lowry, O. H., Rosebrough, N. J. Farr, A.L. & Randall, R. J. Protein measurement with the Foli

phenol reagent. J Biol Chem 193, 265-275 (1951) with th

modifications of Helenius and Simons (Helenius, A. & Simons

K. The binding of detergents to lipophilic and hydrophili

proteins. J Biol Chem 247, 3656-3661 (1972)) using bovine seru

albumin as a standard. The final protein concentrations of th

homogenates should be in a range of 2-4 mg/ml. These dilution were usedfor all analyses.

(s) Statistical Analysis

The relationship between IDH1/IDH2 mutation status and BCAT proteinexpression (FIG. 1 k) was tested with the two-side Fisher's Exact Test.The Student' t test (two-tailed unpaired) was used for all otherstatistical comparisons. *,

<0.05; **, P<0.01; ***, P<0.001.

Example 2 Inhibition of the Cell Proliferation by Gabapentin

The human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB 138) andU373-MG (HTB-17; LGC Standards, Teddington TW11 0LY

U.K.) were cultured in Dulbecco's Minimal Essential Mediu

supplemented with 10% fetal calf serum and 1penicillin/streptomycin-mix. 5×10⁴ cells/well were seeded in 2 wellplates in a total volume of 500 μl cell culture medium The medium wasremoved 4 hours after seeding the cells, an

replaced with 500 μl medium containing1-(Aminomethyl)cyclohexylessigsäure (Gabapentin, dissolved in 200 mMHEPE buffer at a concentration of 500 mM) at final concentrations o

5 mM, 10 mM and 20 mM, respectively. Corresponding volumes o

200 mM HEPES were added to the respective control wells. hours afterexchanging the medium, 5-ethynyl-2′-deoxyuridin

(EdU; Carlsbad, Calif. 92008, USA) was added in order to measur

cell proliferation using Invitrogens (Carlsbad, Calif. 92008, USA ClickiT® proliferation kit. The cells were harvested 16 hour after EdUapplication and proliferation was measured according to themanufacturer's instructions using a BD Biosciences FAC

Canto II flow cytometer (BD Biosciences, Franklin Lakes, N

USA 07417). Three replicate measurements were obtained fo

each treatment. In all cell lines a concentration-dependen

significant reduction of cell proliferation of about 20-55 was observedin the gabapentin treated cells as compared t

mock treated cells. The results are shown in FIG. 10.

Example 3 Inhibition of the Cell Proliferation by BCAT1 AntisenseOligonucleotides

Lentiviral vectors were produced by the cotransfection o

HEK293T cells with the psPAX2 (Addgene 12260, Didier Trono

packaging vector), pMD2.G (Addgene 12259, Didier Trono

envelope plasmid), and pLKO.1 shRNA constructs (Sigma-Aldrich St. Louis,Mo. 63178, USA). Transfections were carried ou

using TransIT®-LT1 (Mirus Bio LLC, Madison, Wis. 53711, USA) an viruswas harvested at 48 and 72 hours after transfection. Tw

independent shRNA constructs targeting different regions o

the BCAT1 mRNA transcript were used: MISSION shRN

TRCN0000005907 NM_(—)005504.3-1064s1c1 (shRNA1) an TRCN0000005909NM_(—)005504.3-751s1c1 (shRNA2); all Sigma Aldrich, St. Louis, Mo.63178, USA. Nontargeting shRNA was use as a control.

The human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB 138) andU373-MG (HTB-17; LGC Standards, Teddington TW11 0LY

U.K.) were seeded in 24 well plates (5×104 cells/well) in total volumeof 500 μl cell culture medium. After 24 hour cells were transduced withvirus in the presence of 8 μg/ml o

polybrene. Virus-containing supernatant was removed after 2 hours andcells were split on day 3 and day 5 afte

transduction. Decreased BCAT1 mRNA and protein expression wa

detected using quantitative real-time PCR and Western blo

(ECA39 antibody, BD Biosciences Pharmingen, San Diego, Calif. 92121,USA). A proliferation assay was carried out on day 6 using the Click-iT®EdU cell proliferation kit after incubating cells with 10 μM EdU for 16hours. In all of the BCAT1 shRNA transduced cells a reduction ofproliferation was observed ranging from 20-80% depending on the cellline. These results are shown in FIG. 11.

Example 4 Determination of BCAT1 Overexpression in IDH^(wt)Glioblastomas

Prediction analysis of microarrays on gene expression data fromastrocytic gliomas of WHO grades II, III and IV identified BCAT1 as thebest classifier distinguishing primary glioblastoma from otherastrocytoma as can be seen in the following Table 3:

Rank Gene symbol AII_AAIII_sGBIV-score pGBIV-score 1 BCAT1 −0.530 0.4352 CHI3L1 −0.503 0.413 3 TIMP1 −0.492 0.404 4 IGFBP2 −0.453 0.372 5 PDPN−0.448 0.368 6 SERPINE1 −0.442 0.363 7 EMP3 −0.436 0.358 8 ADM −0.4130.338 9 PTX3 −0.403 0.331 10 COL6A2 −0.399 0.327 11 NNMT −0.396 0.325 12LIF −0.393 0.323 13 STEAP3 −0.371 0.304 14 COL6A2 −0.371 0.304 15 POSTN−0.361 0.296 16 KCNE4 −0.359 0.295 17 ABCC3 −0.348 0.286 18 FABP5 −0.3430.282 19 LOX −0.342 0.281 20 RANBP17 0.339 −0.278 21 MOXD1 −0.337 0.27622 ADAM12 −0.336 0.276 23 RBP1 −0.331 0.272 24 OCIAD2 −0.330 0.270 25SAA2 −0.320 0.263 26 FMOD −0.319 0.262 27 PBEF1 −0.317 0.260 28 ATP7B−0.316 0.259 29 SOCS3 −0.315 0.259 30 PLAT −0.314 0.258 31 RARRES2−0.312 0.256 32 RAB34 −0.305 0.250 33 VEGF −0.304 0.249 34 RBP1 −0.3030.249 35 SAA1 −0.302 0.248 36 NA 0.300 −0.246 37 PCDH15 0.296 −0.243 38AL354720.14 −0.293 0.240 39 PBEF1 −0.291 0.239 40 EFEMP2 −0.291 0.238 41IL8 −0.288 0.236 42 UPP1 −0.284 0.233 43 ANXA2 −0.282 0.231 44 TNFRSF12A−0.279 0.229 45 ANGPT2 −0.273 0.224 46 ANXA2 −0.272 0.224 47 EMILIN2−0.272 0.223 48 TMEM158 −0.268 0.220 49 VEGF −0.262 0.215 50 PHYHIPL0.262 −0.215

When classifying tumors based on IDH-mutation status, BCAT1 mRNAexpression levels were significantly higher for IDH^(wt) gliomasrelative to IDH^(mut) gliomas (FIG. 1 b; P<0.0001; two-tailed Student'st-test), whereas comparably enhanced levels were not observed for BCAT2expression (FIG. 1 c; P=0.0301). Pathway analysis showed that, inaddition to BCAT1, the RNA expression of several other genes of the BCAAcatabolic pathway was upregulated in IDH^(wt) compared to IDH^(mut)tumors (FIG. 7). Consistent with the RNA expression results, Westernblot analysis showed BCAT1 protein expression was high in IDH^(wt)tumors, but essentially absent in IDH^(mut) tumors, regardless of thespecific mutation in either IDH1 or IDH2 (FIG. 1 d). Sequencing of boththe promoter and coding regions of BCAT1 for 20 gliomas revealed neithernonsense nor putative activating mutations. The observed tightcorrelation between IDH1 or IDH2 mutation and BCAT1 expression wasfurther confirmed through immunohistochemical staining of sections from81 primary human gliomas (77 astrocytoma, 4 oligodendroglioma), amongwhich 45 of 46 IDH^(wt) tumors showed strong BCAT1 staining (FIG. 1 e),while 35 of 35 tumors with mutations in IDH1 (30 R132H, 1 R132C, 1R132S) or IDH2 (3 R172K) were BCAT1 negative (FIG. 1 f-h); (P<0.0001;Fisher's Exact Test; FIG. 1 k). The BCAT1 staining pattern therefore islargely complementary to the pattern obtained with the widely usedantibody against the IDH1-R132H mutant protein (FIG. 1 i,j). However,unlike IDH1-R132H staining, the BCAT1 antibody also distinguishesIDH^(wt) tumors (FIG. 1 e) from tumors with less common IDH1 and IDH2mutations that are not recognized by anti-IDH1-R132H (FIG. 1 g-h). Thesedata show that high BCAT1 expression is a characteristic feature ofIDH^(wt) gliomas that can be used to positively identify these tumors ina diagnostic setting.

Example 5 Determination of Substrate-Dependent Expression of BCAT1

BCAT1 was found to be expressed strongly in the glioblastoma cell linesLN-229, U-87MG, U-373MG and to a lesser extent in A172 (FIG. 2 a), allof which were confirmed to carry IDH1 and IDH2 wildtype genes. BCAT1expression also was elevated in the Hs683 cell line, which wasoriginally derived from an oligodendroglioma but nevertheless displaysan IDH^(wt) genotype. Thus, all these cell lines can be consideredsuitable models for studying BCAT1 function. BCAT1 RNA and proteinexpression were upregulated under hypoxic conditions, which arefrequently present in glioblastoma (FIG. 2 b). BCAT1 expressioncorrelated with the concentration of the substrate alpha-KG and wasupregulated after Increasing the concentration of cell-permeabledimethyl-alpha-KG-substrate in the culture medium (FIG. 2 c).Conversely, shRNA-mediated knockdown of IDH1, a major source of alpha-KGin the cytoplasm, led to strong downregulation of BCAT1 expression (FIG.2 d).

Example 6 Differentially Regulated BCAT1 Expression ThroughDNA-Methylation in Two Alternative Promoters

To further elucidate the differential regulation of BCAT1 expression inIDH^(wt) and IDH^(mut) gliomas, transcript expression in patient sampleswas quantified. Using RT-PCR and sequencing, expression of threeprotein-coding BCAT1 transcripts listed in the Ensembl database inIDH^(wt) astrocytic primary tumors as well as in a pool of 23 normalbrain tissues (FIG. 9) were confirmed. These three transcripts (T1, T4and T6) encode proteins of 386, 398, and 385 amino acids, all three ofwhich correspond to the single protein band of 43 kD as identified byWestern blot analysis. These transcripts originate from two alternativepromoters (FIG. 3 a) and differ only in their first exons, which encode2, 14 and 1 amino acid(s) in T1, T4 and T6, respectively.Transcript-specific qRT-PCR identified T6 as the predominant transcriptrepresenting 73% of all BCAT1 mRNA in primary tumors (FIG. 3 b).Notably, expression of all BCAT1 transcripts was significantly higher inIDH^(wt) compared to IDH^(mut) tumors.

To investigate the possible mechanisms of BCAT1 transcriptionalregulation, quantitative DNA methylation analysis on astrocytomas of allgrades using MassARRAY analysis of PCR products amplified frombisulfite-treated DNA covering the two alternative promoters (FIG. 3 c)were performed. Distinct methylation patterns were observed for IDH^(wt)and IDH^(mut) tumors. The major promoter (promoter 2) washypermethylated in IDH^(mut) tumors but mostly unmethylated in IDH^(wt)tumors and normal brain (FIG. 3 c, right panel). The average degree ofmethylation of the A6 and A7 amplicons was strongly associated with IDH1mutation status (FIG. 3 d). These data show the suppression oftranscripts T4 and T6 by promoter-2 methylation in IDH^(mut) tumors.Promoter 1 (FIG. 3 c, left panel) was mostly unmethylated in all tumorsamples as well as in normal brain, with the exception of a stretch ofhypermethylated sequences immediately upstream of the CpG island. Withinthis upstream region, three differentially methylated CpG-dinucleotidesbetween IDH^(wt) and IDH^(mut) tumors at positions −699/−697 (CpG4;5)and −660 (CpG6) in amplicon A2 were identified. In contrast to themethylation pattern in promoter 2, CpG4;5 (FIG. 3 e) and CpG6 (FIG. 3 f)showed significantly less methylation in IDH^(mut) than in IDH^(wt)tumors. The degree of methylation in this differentially methylatedregion (DMR) correlated with the expression of the BCAT1 transcript T1in IDH^(wt) tumors supporting its functional relevance (FIG. 3 g). Theobserved CpG-specific methylation pattern is consistent with repressorbinding to the DMR leading to the downregulation of T1. Binding of therepressor HEY1 to the BCAT1 promoter 1 but not promoter 2 (FIG. 3 a)previously had been suggested by ChIPseq data. Analysis of RNAexpression data confirmed overexpression of the HEY1 repressor inastrocytic tumors compared to normal brain. Consistent withHEY1-repressor activity, siRNA-mediated HEY1 knockdown in HEK293T cellsincreased transcript-T1 expression (FIG. 3 h,i). ChIP analysisdemonstrated that compared to an upstream control region and to a regionclose to the translation start site, the strongest binding of twodifferent HEY1 constructs occurs in the DMR (FIG. 3 j,k). Together,these data strongly show differential expression of BCAT1 transcripts inastrocytomas is regulated by DNA methylation involving broad methylationof promoter 2 in IDH^(mut) tumors and CpG-site specific methylation in aHEY1-repressor binding site in promoter 1 of IDH^(wt) tumors.

Example 7 Reduction of the Release of Glutamate by Glioblastoma CellsThrough the Suppression of BCAT1

To gain insight into the BCAT1 functional role in glioblastomas, celllines U-87MG and U-373MG cell lines were treated with gabapentin, aleucine analog that specifically inhibits BCAT1, but not BCAT2. ¹H-NMRspectroscopy of extracts of cells treated with 20 mM gabapentin for 20hours demonstrated the intracellular accumulation of BCAAs, consistentwith BCAT1 inhibition (FIG. 4 ab). Glioblastoma release highconcentrations of glutamate which leads to neuronal death by anexcitotoxic mechanism. Glutamate release was significantly reduced withinhibition of BCAT1 with gabapentin (FIG. 4 c) indicating that BCAT1 isa major contributor to glutamate production through BCAA catabolism inIDH^(wt) glioma.

To independently confirm these findings, shRNA-mediated BCAT1 knockdownin U-87MG, U-373MG and Hs683 cells using two shRNAs that both target allthree BCAT1 transcripts was performed. Tandem-MS analysis of the U-87MGcell culture media after 24 hour incubation revealed that glutamaterelease, as well as BCAA uptake were reduced in BCAT1 knockdown cellscompared to control cells (FIG. 4 d). A significant increase in releaseof alanine, glycine and threonine was also observed. Quantification ofintracellular amino acid concentrations revealed significantaccumulations of aspartate, glycine and threonine (FIG. 4 e). A smallbut significant accumulation of glutamate also was observed followingBCAT1 knockdown; however, considering the high ratio of media volume tointracellular volume, total concentration of glutamate is notsignificantly affected by this small intracellular accumulation. In theBCAT2 knockout mice the inhibition of BCAA catabolism results in highconcentrations of BCAAs and higher rates of protein synthesis inperipheral tissues via U) the mechanistic target of rapamycin (mTOR)signaling pathway and compensatory increased protein degradation(increased protein turnover).

BCAT1 knockdown led to strong downregulation of 3-hydroxyacyl CoAdehydrogenase (HADH), an enzyme participating in the catabolism ofvaline and isoleucine downstream of BCAT1 (FIG. 4 f, g). Since HADH iscentral to fatty acid metabolism, BCAT1 knockdown would also altersynthesis or degradation of fatty acids essential for membranesynthesis.

Example 8 Limitation of Glioblastoma Migration in Microchannels by BCAT1Knockdown

BCAT1 knockdown strongly affected cell morphology, resulting in arounded, less extended appearance (FIG. 5 a-b). To test whether thesemorphology changes could affect the tumor cells' ability to invadeadjacent tissues, a microchannel migration chip to simulate athree-dimensional environment was used (FIG. 5 c). Following BCAT1knockdown, the majority of U-87MG cells (55%) penetrated short distancesinto the microchannels, but were unable to actively deform themselves inorder to completely invade the microchannels whereas most control cells(78%) completely invaded the channels (FIG. 5 d). This reducedinvasiveness of the BCAT1 knockdown cells might be due to alteredmembrane composition caused by the observed lower abundance oflong-chain fatty acids and differences in cholesterol metabolism (FIG. 4g); such changes might sufficiently affect general availability ofmembrane components as well as membrane elasticity to hinder cellinvasion.

Example 9 BCAT1 is Essential for Glioblastoma Growth

To assess the impact of BCAT1 on tumor cell proliferation, inhibitionand knockdown experiments were conducted. A concentration-dependentreduction of proliferation up to 56%, estimated based onEdU-incorporation, was observed, upon treatment with the inhibitorgabapentin (FIG. 6 a). Cell cycle analyses suggested that gabapentintreatment induced partial G1-arrest, indicated by the increased fractionof cells in G1-phase with concurrent decreases of the G2 and S-fractions(FIG. 6 b). ShRNA-mediated BCAT1 knockdown elicited similar effects(FIG. 6 c,d). BCAT1 knockdown decreased proliferation by 20-70% in allthree cell lines (FIG. 6 c) and led to G1-arrest and strong increases incellular CDKN1B/p27^(KIP1). Notably, the degree of CDKN1B/p27^(KIP1)accumulation showed a positive correlation with the size of theG1-fraction (FIG. 6 d). Cell death, indicated by the fraction of cellsin sub-G1 phase, remained below 5% except in the case of Hs683, whichshowed a moderate increase as shown in the following Table 4

Cell line Treatment Average sub-G1 [%] STDEV sub-G1 U87-MG nt shRNA 2.10.241 BCAT1 shRNAI 1.4 0.100 BCAT1 shRNAII 1.9 0.058 20 mM Gabapentin2.4 0.354 Control (HEPES) 1.4 0.707 U373-MG nt shRNA 2.4 0.283 BCAT1shRNAI 1.1 0.045 BCAT1 shRNAII 2.7 0.141 20 mM Gabapentin 2.8 0.283Control (HEPES) 1.4 0.212 HS683 nt shRNA 13.3 1.344 BCAT1 shRNAI 21.40.424 BCAT1 shRNAII 16.7 2.263 20 mM Gabapentin 10.1 0.849 Control(HEPES) 2.2 0.424

Comparable reductions of cell proliferation were observed upon BCAT1knockdown in glioblastoma primary spheroid cultures in serum-free media,except that these cells showed a higher rate of apoptosis as determinedby Annexin V/7AAD staining (FIG. 9). Consistent with the observedreduction in proliferation, BCAT1 knockdown led to decreasedphosphorylation of the v-akt murine thymoma viral oncogene homolog (AKT)in U-87MG, U-373MG and Hs683 cells (FIG. 6 e).

The effect of BCAT1 knockdown on tumor growth in vivo was evaluated byintracerebral transplantation of U-87MG cells into CD-1 nude mice (FIG.6 f-h). Four weeks after transplantation of equal numbers of livingcells, all 6 control mice, but only 1 of 6 mice transplanted withBCAT1-shRNA-transduced cells displayed neurologic symptoms such aslethargy or uncoordinated motor activities. Hematoxylin and eosinstaining of mouse brain sections revealed large tumors in micetransplanted with control cells (FIG. 6 f) while significantly smallertumors were found in mice transplanted with BCAT1-knockdown cells (FIG.6 g). Quantitative analysis confirmed significant differences in tumorvolume between the groups (FIG. 6 h, P=0.0091).

1.-12. (canceled)
 13. A pharmaceutical composition for treating a braintumor, wherein said pharmaceutical composition comprises an effectivedose of a compound capable of reducing or inhibiting (a) the biologicalactivity of a branched-chain-aminotransferase-1 (BCAT1) or (b) theexpression of the gene encoding BCAT1, wherein the effective dose leadsto a reduction or inhibition of the proliferation of brain tumor cellsin a subject, and a pharmaceutically acceptable carrier.
 14. Thepharmaceutical composition of claim 13, wherein said compound is anantisense oligonucleotide or siRNA reducing or inhibiting the expressionof the gene encoding BCAT1.
 15. The pharmaceutical composition of claim13, wherein said compound is a compound reducing or inhibiting thebiological activity of BCAT1.
 16. The pharmaceutical composition ofclaim 15, wherein said compound is 1-(aminomethyl)cyclohexaneaceticacid.
 17. The pharmaceutical composition of claim 15, wherein saidcompound is an antibody directed against BCAT1 or a fragment thereof.18. The pharmaceutical composition of claim 13, wherein said compound isan inactive version of BCAT1.
 19. The pharmaceutical composition ofclaim 13, wherein said compound is, wherein the neoplasm to be treatedshows BCAT1 (over) expression.
 20. The pharmaceutical composition ofclaim 13, wherein said compound is, wherein said brain tumor to betreated is an astrocytic brain tumor, glioma or glioblastoma.
 21. Thepharmaceutical composition of claim 20, wherein said compound is,wherein the brain tumor is an IDHwt glioblastoma.